1. Field of the Invention
The present invention relates to a method of and an apparatus for measuring a target substance contained in a mixture sample, particularly a vital substance such as urine or blood plasma, by a light scattering method for qualitatively and quantitatively measuring its component such as glucose, acetone or urea, and more particularly, it relates to a measuring method and a measuring apparatus employing anti-Stokes-Raman scattering as light scattering.
2. Description of the Background Art
There are many methods of quantitatively measuring concentrations of substances by light scattering. In relation to methods employing Raman scattering, there is a method of nondestructively measuring a concentration through the fact that Raman scattering intensity is proportionate to the glucose concentration in a sample (refer to Japanese Patent Laying-Open Gazette No. 7-49309 (1995)). This method utilizes near infrared light as excitation light for suppressing fluorescence from the sample, condenses scattered light from the sample by a lens, blocks off Rayleigh scattered light through a notch filter, and thereafter separates the scattered light into its spectral components through a spectroscope for obtaining the Raman scattering spectrum of a target substance contained in the sample, thereby determining the target substance.
In general, a laser of near infrared or a longer wavelength is employed in order to determine a target substance while avoiding fluorescence generated from a mixture of a substance derived from an organism or another fluorescing substance ("Laser Raman Bunko to Rinsho Igaku" by Yukihiro Ozaki et al., O Plus E 1992 4 92-99). However, the intensity of Raman scattering is weakened in inverse proportion to the fourth power of the excitation wavelength, and hence the intensity of detected light is remarkably weakened if near infrared light is employed as the excitation light, as compared with excitation by a visible light.
If the excitation light has a longer wavelength than visible, a detector and a spectroscope must be rendered suitable for a longer wavelength. Near infrared surface and array devices are extremely high-priced at present. While methods of measuring Raman scattering through near infrared wavelength light include FT-Raman spectrometry employing Fourier transformation, an FT-Raman spectroscope is large-sized and disadvantageously takes a long time for measurement.
When a sample is measured through the aforementioned Raman scattering or light scattering, the target scattered light spectrum from the sample is often vanished by fluorescence from the sample. The fluorescence is particularly intense when the sample is an organism itself, a substance such as blood, urine, excrements, saliva or tear separated and collected from an organism, or food, fruit or farm products.
In Raman scattering spectra, spectra in regions having large and small photon energy values with respect to excitation light respectively (these spectra are hereinafter referred to as anti-Stokes and Stokes lines respectively) are observed. The Stokes line appears since small parts of molecules holding photons return not to original vibration levels but to vibration levels having different electron ground states (levels higher than the ground states) after liberating the held photons when specific molecules are irradiated with light. On the other hand, the anti-Stokes line appears since electrons originally present at levels having higher energy than ground states do not return to the original levels but make transition to ground state levels along with parts of applied photons after liberating most parts of the applied photons. Namely, the energy widths of the anti-Stokes and Stokes lines become energy differences between the ground states and vibrational excitation states, and hence the anti-Stokes and Stokes lines generally appear on symmetrical positions in terms of shift wavenumbers with respect to the excitation light.
In the fluorescence, on the other hand, a spectrum is present in a region having small quantum energy with respect to the excitation light, i.e., a region having a long wavelength, unless excitation and fluorescence spectra remarkably intersect with each other. Quantumly considering this, the fluorescence is generated since electrons which are present in ground states or at other levels are excited by applied energy through the energy of applied light and stay at a number of levels for an instance during transition from the excitation energy levels to the ground states. Namely, the fluorescence is generated in a region smaller (longer wavelength side) than the quantum energy of the excitation light in general.
Therefore, it is conceivable that a component spectrum of a target substance can be obtained from a sample by a method of Raman spectral analysis while avoiding influence by fluorescence when the anti-Stokes line in Raman scattering analysis is observed, and identification and determination of the target substance can be performed by observing change and intensity of the spectrum of the target substance.
Study of anti-Stokes-Raman scattering includes coherent anti-Stokes-Raman spectroscopy (CARS) for introducing pump light and probe light into a target substance and measuring anti-Stokes-Raman scattering. In the principle of this spectroscopy, wavelengths of two different vibration numbers are introduced into a sample and spectra are forcibly oscillated by a transition vibration number of Raman scattering when the quantum energy difference of the incident light coincides with the transition vibration number of Raman scattering for utilizing the fact that forced vibration in the interior of the substance generates phased nonlinear vibration. In an anti-Stokes-Raman spectrum by CARS, therefore, nonlinear interaction of the light and the substance becomes remarkable dissimilarly to the relation between a concentration and Raman scattering intensity of the target substance in an ordinary linear Raman scattering spectrum.
Further, nonlinear phenomenons such as multiple photon absorption transition, scattering by a higher-order coherent Raman process, induced Raman scattering, a sum frequency and higher harmonic generation coexist, and the spectrum does not necessarily linearly reflects the concentration of the target sample. Therefore, CARS is not utilized as a quantitative analysis method.
Further, many laser light source units are necessary for a spectroscopic system, and hence the apparatus becomes large-scale.
The intensity ratio of anti-Stokes-Raman and Stokes-Raman scattering spectra is approximated by the following equation of Boltzmann distribution:
anti-Stokes intensity/Stokes intensity=exp (-h.nu./kT) where h represents the Planck's constant, k represents the Boltzmann's constant, T represents the absolute temperature, and .nu. represents the Raman shift wavenumber.
FIG. 1 shows calculated values of intensity ratios of anti-Stokes-Raman and Stokes Raman lines on the assumption that the absolute value is 300 K on the basis of the Boltzmann distribution. When the sample temperature is 300 K, the intensity ratio is 0.008 at -1000 cm.sup.-1 (minus sign of the wavenumber expresses an anti-Stokes-Raman scattering by shifting toward a shorter wavelength side than the excitation wavelength), and 0.007 at -1500 cm.sup.-1. Therefore, it has generally been considered impossible to detect anti-Stokes-Raman scattered light by ordinary linear Raman spectroscopy.